Abstract
Purpose :
Elevated levels of transforming growth factor beta 2 (TGFβ2) drive a myofibroblast-like Trabecular Meshwork TM phenotype that has been associated with fibrosis and elevated outflow resistance in primary open-angle glaucoma (POAG). It is unclear whether this process involves dysregulation of TM pressure sensing and transduction, and what the molecular mechanisms that integrate biochemical (TGFβ) and biomechanical (IOP) glaucoma risk factors might be. We investigated the mechanistic relationship between a known TM mechanosensor, TRPV4 (transient receptor potential vanilloid 4), and TGFβ2 signaling in cultured cells and POAG mouse models to ascertain the functional significance of the interaction between TM pressure sensing and TGFβ2 signaling.
Methods :
Quantitative reverse-transcription PCR (qRT-PCR), Western blot, and fluorescent calcium imaging were used to profile the response of primary human and mouse trabecular meshwork cells to TGFβ2 and to assess the functionality of mechanosensitive ion channels in-vitro. Contractility measurements were conducted using biomimetic TM-seeded gels. Diurnal IOP measurements were used to assess ocular hypertension in a lentiviral TGFβ2 overexpression model.
Results :
Chronic TGFβ2 treatment significantly increased TRPV4 expression, membrane insertion, and augmented TRPV4 agonist-induced Ca2+-influx (258% of control). TGFβ2 and TRPV4 treatments independently upregulated cellular actin, but TM contractility was inhibited by treatment with a TRPV4 antagonist, and hypercontractility was transiently induced by application of a selective TRPV4 agonist. Lentiviral TGF-β2 overexpression significantly increased IOP in WT C57 mice, but not in TRPV4-/- mice. Intracameral injection of a TRPV4 antagonist in WT TGF-B2 overexpressed mouse eyes transiently lowered IOP to that of LV-Ctrl WT eyes.
Conclusions :
These experiments suggest a central role for dynamic mechanochannel signaling in TGFβ2-induced ocular pathology. In vivo, TM cells may undergo transdifferentiation due to overactive TGFβ2 signaling, yet even fibrotic TM tissue preserves, or even potentiates, pressure-dependent activation of transducers required to maintain ocular hypertension. Our pharmacological treatment and data from genetically modified animals show that mechanochannel targeting could represent a viable method to mitigate growth factor-induced ocular hypertension.
This abstract was presented at the 2024 ARVO Annual Meeting, held in Seattle, WA, May 5-9, 2024.